Testicular torsion is a medical emergency which can cause permanent loss of spermatogenesis. The lesion occurs most commonly in adolescent and pre-adolescent males, and it is acknowledged that the torsion must be diagnosed and treated promptly to avoid the loss of function of the affected testis. Previous studies of the vascular and cellular responses to testicular torsion in the rat model have indicated three separate but related mechanisms of tissue injury. Altered microvascular blood flow, production of intratesticular torsion reactive oxygen species, and induction of germ-cell apoptosis. The specific aims in this application will test the overall hypothesis that these three are related in the development of testicular pathology. The testis become ischemic during torsion, but microvascular flow patterns do not return to normal after relief of torsion. We propose to determine the role of neural and non-neural vasoregulation in this alteration. Reperfusion after an ischemic period causes the release of reactive oxygen species (ROS) in other tissues, and ROS can damage cell membranes through lipid peroxidation or can stimulate apoptosis, among other effects. We propose to determine if ROS play a role in the mechanism of the tissue injury seen after relief of testicular torsion, and we propose to determine if ROS induce apoptosis in germ cells. Preliminary evidence indicates that torsion does induce apoptosis specifically in germ cells. We not only propose to determine if the mechanism initiating apoptosis involves ROS, but we propose to partially dissect the molecular pathway to the apoptosis induced by torsion. We will identify key molecules in the germ cell apoptotic pathway by utilizing a gene knock-out strategy in mice. Such an approach utilizes strains of mice lacking the gene for specific proteins in the apoptotic pathway. The germ cell reaction to torsion in these strains will tell whether or not the particular protein under study is important in the pathway. Our studies of both ROS and apoptotic pathway proteins have potential to lead to improved therapies for human patients.